Apparatus for sample preparation

ABSTRACT

The invention, in various embodiments, provides systems, methods and devices relating to processing a sample.

REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. provisional application Ser.No. 60/528,069, filed Dec. 8, 2003, the disclosure of which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention generally relates to systems, methods and devices forpreparing and processing samples. More specifically, in variousembodiments, the invention relates to pulverizing and/or fragmentingsamples to ready them for analysis.

BACKGROUND

A first step in sample analysis typically involves collecting thesample. For example, a first step in a biological analysis such as RNAgene expression profiling or protein biomarker profiling is to collect aparticular sample so that its biochemical constituents can be analyzed.However, prior to analysis, a solid sample specimen, typically, isprepared by deconstructing it into a plurality of smaller fragments ofthe specimen to enable more accurate analysis.

Recently, downstream analytical processing of samples has undergonesignificant improvements, including with regard to sensitivity,throughput and like. Due to these significant improvements in downstreamprocesses, deficiencies in upstream sample collection and preparationhave become more apparent. One upstream processing enhancement has beenthe ultrasonic systems and methods for treating a sample described inco-pending, co-owned U.S. patent application Ser. No. 10/777,014,entitled Apparatus and Methods for Controlling Sonic Treatment,” theentire disclosure of which is incorporated by reference.

A challenge of sample preparation is that the types of samples arediverse. For example, samples may be biological, non-biological or acombination thereof. They may be from animals or plants. Samples mayinclude, without limitation, cells, tissues, organelles, bones, seeds,chemical compounds, minerals, metals, or any other material for whichanalysis is desired.

Sample preparation is particularly challenging for solid biologicalsamples, such as tissue samples. Physical and/or chemical approaches areoften employed to disrupt and homogenize the solid sample forbiochemical extraction. While appearing deceptively simple,transitioning a sample of biologically active tissue, for example, onthe order of 1 gram, to a plurality of biomolecules that are stabilizedand isolated in an appropriate analytical solution is exceedinglycomplex, very difficult to control, and prone to introduction of errorsand/or sample constituent degradation.

Another challenge associated with sample preparation relates to thelability of the target molecules. For some applications, an overridingcriterion is to retain the native biochemical environment prior tosample collection and throughout the extraction process, withoutperturbing the biochemical constituents to be analyzed. For example,RNases are extremely robust and may significantly degrade the mRNAprofile of a tissue sample if the RNases are not immediately stabilized(typically thermal or chemical inactivation) at the time of tissuecollection and during sample processing or homogenization. Often, tominimize perturbation of the biochemical profile of the sample, thetissue is flash-frozen (e.g., via direct immersion of the samplefollowing procurement in liquid nitrogen) and stored at cryogenictemperatures (e.g., −80° C. or lower), which inhibits degradativeprocesses.

Conventionally, once a sample is stabilized from thermal and/or chemicaldegradation, it is pulverized in liquid nitrogen at a temperature ofabout −196° C., for example, using a mortar and pestle. Otherpulverizing systems available include a rotor-stator (polytron) and abead-beater apparatus, which do not operate at cryogenic temperatures.In a typical example, a frozen specimen having a volume of approximately1 cm³ may be fragmented into a plurality of solid fragments each havinga volume of approximately 100 um³ or less.

Prior art approaches for performing such fragmentation suffer from manydrawbacks. One such drawback is that liquid nitrogen is difficult andhazardous to work with. Another drawback is that prior art approachescan be slow and tedious. A further drawback is they involve directcontact between the sample and the fragmenting agents. For example, thesample, typically, is not contained during fragmentation causingportions of the sample to be deposited on the fragmenting devices in anon-recoverable manner. This, in turn, causes reduced sample recoveryand extensive apparatus cleaning between operating cycles. Also, suchdeposits cause increased operator exposure to potentially hazardoussamples, and/or the sample may be degraded by enzymes, bacteria, fungi,or other external contaminants.

Another challenge to sample preparation is maintaining the sample at anappropriate temperature. A disadvantage of the direct contact prior artdevices, particularly the automated prior art devices, is that thesample may become sufficiently heated to cause the sample to degrade.This disadvantage is accentuated by sample fragmentation due toincreased thermo-sensitivity resulting from increased surface area.Thawing also makes it difficult to transfer sample particles to a vesselfor further processing. Another drawback is that conventional techniqueshave size range limitations. For example, a high percentage of a 25-mgsample would be lost in a 5-ml bead-beating system, causing unacceptablylow sample recovery.

Accordingly, there is a need for an improved approach to preparingsamples for further analysis.

SUMMARY OF THE INVENTION

The invention addresses the deficiencies of the prior art by providing,in various embodiments, systems, methods and devices for collecting,stabilizing, fragmenting and/or analyzing samples. As described above,analysis of biological and non-biological sample specimens often beginswith collection of a sample of relatively large size. Before theconstituents of such a sample can be effectively analyzed, the sample,preferably, is fragmented into a plurality of smaller specimens. Suchsmaller specimens can then be stored, analyzed, or further processed.

A sample may be any material. Exemplary samples include, but are notlimited to, bones, teeth, seeds, plants, pathological ornon-pathological animal tissue (e.g., muscle, liver, kidney, lung,brain, pancreas, prostate, ovary, breast, etc.), tumor tissue, rocks,mineral samples, tree bark, and/or food products. Exemplary constituentsinclude, but are not limited to, nucleic acids, amino acids,polypeptides, bacteria, viruses, fungi, spores, small organic molecules,small inorganic molecules, metals, minerals, ores, and the like. Thesample may be relatively soft, such as a tissue sample, may berelatively hard, such as a bone or mineral sample, and may include sharpknife-like edges and/or sharp needle-like points.

In one aspect, the invention provides a sample vessel for containing asample. According to one embodiment, the sample vessel includes achamber portion and a port portion. According to one feature, thechamber portion provides a structural barrier between the sample and anexternal environment. According to another feature, the barriermaintains the sample in a sterile environment. In one embodiment, thesample vessel is formed from a film material. According to oneconfiguration, the film material is formed into a bag for containing thesample. The bag may be formed by folding a single sheet of the film andbonding it together on at least two sides. Alternatively, the bag may beformed from two sheets of film bonded together on at least 3 sides. Inother configurations, the vessel is entirely, or includes portions thatare, injection molded. In various embodiments, the sample vessel isformed, at least in part, from a polyimide, polysulfone, liquid crystalpolymer, fluorinated polymer, and/or other like material. According toone embodiment, the bag is formed from one or more sheets of Kapton™material (part number 150FN019 available from Dupont).

According to one feature, the sample vessel includes an aperture of anytype suitable port for introducing and/or removing a sample and/or othermaterials from the chamber. In various configurations, the port isformed from a substantially rigid, substantially hard, and substantiallynon-deformable material, such a polypropylene or other suitable polymerplastic. Preferably, the port includes a hub fitting and a cap or coverfor snap, pressure, screw, or other reversible sealing mating with thehub fitting to provide an additional barrier between the sample and anexternal environment. In some embodiments, the port is sized and shapedfor mating with a second vessel for transferring the sample and/or othermaterials from the sample vessel to the second vessel. In otherconfigurations, the port may configured for reversibly mating with asyringe for injecting and/or removing the sample and/or other materialsinto or out of, respectively, the sample vessel. According to one suchconfiguration, the port is configured for conventional needleless matingwith a syringe, for example, such as the needleless ports employed fortransfer of blood products. In another such configuration, the portincludes a diaphragm for resealable puncture by a syringe needle. Insome implementations, a single aperture serves as the aperture formating with the transfer container and the aperture for introducing thesample. In other embodiments, separate apertures are employed.

In one embodiment, the sample vessel includes a portion flexible enoughto deform nondestructively (e.g., without experiencing cracking,tearing, ripping or other degradation in structural integrity), or insome embodiments substantially nondestructively, in response to amechanical impact sufficient to fragment the particular sample containedwithin the sample vessel. According to one feature, subsequent to themechanical impact, the sample vessel continues to maintain sufficientstructural integrity to continue to separate the sample from theexternal environment. In one embodiment, the vessel continues tomaintain the sample in sterile isolation from the external environment.According to various implementations, the mechanical impact may have animpact energy transfer of greater than or equal to about 1 Joule, 2Joules, 3, Joules, 4 Joules, 5 Joules, 6 Joules, 7 Joules, 8 Joules, 9Joules, or 10 Joules. According to one implementation, the mechanicalimpact may occur with the sample at cryogenic temperatures below about−20° C., −30° C., −40° C., −50° C., −60° C., and/or −70° C. In someimplementations, the exposure to the mechanical impact may occur withthe sample at cryogenic temperatures between about −80° C. and about−196° C. According to another feature, the vessel is for single use.According to another feature, the vessel may be substantially evacuated.

Depending on the mechanical properties of the sample (e.g., relativelyhard, relatively soft, forms sharp or pointed shards when fragmented,etc.) and the temperature at which fragmentation is to occur, variousmaterials may be suitable for the vessel. For example, a brain samplemay require a particular film layer (e.g., 1 mil). Alternatively, abone, seed, or rock sample, which may have sharp and or pointedfeatures, may require a thicker film layer (e.g., 4 mil), an additionalreinforcement layer, for example, of a non-woven polymer material, suchas Tyvek™ (available from Dupont), reinforcement by woven or non-wovenmaterial, or other suitable reinforcement.

In various embodiments, the sample vessel is formed from a range ofmaterials and a range of wall thicknesses to produce sample vesselsappropriate for a variety of uses. For example, the sample vessel may beconstructed from materials compatible for use at the above describedcryogenic temperatures, as well as materials compatible for use only atnon-cryogenic temperatures, such as room temperature and temperaturesabove the freezing point of water. By way of further example, the one ormore walls of the vessel may be approximately 0.5-5 mil thick. Exemplarywall thicknesses include, but are not limited to, about 0.75 mil, 1 mil,1.25 mil, 1.5 mil, 1.75 ml, 2 mil, 2.25 mil, 2.5 mil, 2.75 mil, 3 mil,3.25 mil, 3.5 mil, 3.75 mil, and 4 mil.

According to some configurations, the sample vessel includes a substratehaving a chamber for containing the sample formed thereon (e.g., ablister pack), wherein the chamber includes a portion flexible enough tonondestructively deform in response to a mechanical impact sufficient tofragment the sample contained within the chamber. In otherconfigurations, the sample vessel includes one or more resilientlyflexible walls.

According to some embodiments, the sample vessel includes at least twosubstantially rigid walls connected along their perimeters by one ormore walls flexible enough to nondestructively deform sufficiently toallow the at least two substantially rigid walls to come together andcontact each other with sufficient force to fragment the sample inresponse to a mechanical impact on at least one of the substantiallyrigid walls. In one implementation, the flexible walls are formed asaccordion-like structures that fold along preformed creases in responseto the mechanical impact.

In various embodiments, the sample vessel is sized and shaped forinsertion into and functional interoperation with a mechanical impactproviding device. In other embodiments, the sample vessel is also oralternatively sized and shaped for insertion into and functionalinteroperation with a sample preparation device for providing focusedacoustic energy to the sample contained within the vessel for performingany one of: cooling; heating; fluidizing; mixing; stirring, disrupting,increasing permeability of a component of, enhancing a reaction of,sterilizing; and/or further fragmenting the sample. Such a samplepreparation device is described in co-pending, co-owned U.S. patentapplication Ser. No. 10/777,014, entitled Apparatus and Methods forControlling Sonic Treatment,” the entire disclosure of which isincorporated by reference above.

In one embodiment, the vessel includes a mechanical, optical, and/orelectronic security feature configured for interacting with acorresponding security feature on the mechanical impact providing deviceand/or the acoustic energy providing device, without which interaction,the mechanical impact providing device, and/or the acoustic energyproviding device, respectively, will not function.

According to another aspect, the invention provides a kit including asample vessel having any of the above described features, and a secondvessel for mating with the sample vessel as described above. In oneembodiment, the kit includes a suitable agent, reagent, buffer or thelike for combining with the sample subsequent to fragmentation.According to one feature of this embodiment, the agent, reagent, bufferor the like is prefilled into the second vessel. According to anotherfeature of this embodiment, the second vessel includes a barrier formaintaining the agent, reagent, buffer or the like separated from thechamber of the sample vessel until after fragmentation of the sample. Inone embodiment, the barrier is broken by rotating or otherwisemanipulating the sample vessel relative to the second vessel, to enablethe agent, reagent, buffer or other material to fall, flow and/or befunneled into the sample vessel, or to enable the fragmented sample tofall into the second vessel.

According to another aspect, the invention is directed to a device forproviding a mechanical impact to a sample vessel. In one embodiment, themechanical impact device includes a base, a holder into which the vesselcan be interfitted, and at least one impact surface for impacting thesample vessel. The impact surface can be driven, for example, by ahammer, solenoid, pneumatically actuated device, hydraulically actuateddevice, gravity actuated device, or any other suitable mechanism.According to one embodiment, the device includes a processor forcontrolling operation of the at least solenoid. According to onefeature, the mechanical impact device includes a user-adjustable controlfor adjusting the impact force provided by the at least one solenoid.The user-interface may also include a temperature adjustment forselecting a temperature at which the impact providing device is to bemaintained. According to another feature, the mechanical impact deviceincludes a chamber/well for receiving an immersion chiller probe formaintaining at least the portion of the device containing the samplevessel at a lowered temperature to help maintain the sample at asuitable temperature to avoid sample degradation. According to anotherfeature, other portions of the device, particularly those contacting awork surface on which the device may be placed, are maintained near orat room temperature. According to a further feature, the mechanicalimpact device includes a mechanical, optical and/or electronic securityfeature configured to interact with a corresponding security feature onthe sample vessel, without which interaction, the mechanical impactproviding device will not function.

According to another aspect, the invention is directed to a system forfragmenting a sample specimen, the system including a sample vessel forcontaining a sample specimen, and a mechanical impact providing devicefor receiving the sample vessel into an impact zone. In one embodiment,the vessel includes a portion flexible enough to deform nondestructivelyin response to a mechanical impact from the mechanical impact providingdevice sufficient to fragment a sample specimen contained within thesample vessel into a plurality of smaller sample specimens.

According to a further aspect, the invention provides a method forpreparing a sample for analysis, storage or further processing. Themethod includes the steps of, placing a sample into a sample vessel,applying a mechanical impact to an external surface of at least aportion of the sample vessel having sufficient force to fragment theparticular sample, while maintaining structural integrity of theenclosed vessel during application of the mechanical force. According toone feature, the method includes maintaining the sample at a temperaturebelow about −20° C., −30° C., −40° C., −50° C., −60° C., and/or −70° C.during application of the mechanical force. According to anotherfeature, the method includes maintaining the sample at a temperaturebetween about −80° C. and about −196° C. during application of themechanical force. According to a further feature, the method includesstoring the sample in the sample vessel subsequent to fragmentation forfuture analysis.

In an alternative embodiment, the impact providing device operates at orabout room temperature. However, the impact providing device operatesquickly enough so that the transient exposure to the impact surfaces donot substantially warm the sample. This is also the case where thesample, itself, is maintained at room or near room temperature. Invarious embodiments, the impact providing device may provide elementsfor heating or cooling the sample prior or subsequent to fragmenting it.

According to one process, the method includes transferring the samplefrom the sample vessel to a mated second vessel subsequent tofragmentation. According to various embodiments, the method includessubjecting the sample to a focused acoustic field while contained in thesample or second vessel, subsequent to applying the mechanical impact.

According to an additional aspect, the invention is directed to systemsand methods for processing a sample. According to one embodiment, thesystems and methods include employing an automated vessel handling robotfor retrieving a sample vessel of the type described herein from astorage location, providing it to an impact providing device forfragmenting the sample, inverting the sample vessel to transfer thefragmented sample into a second vessel, adding an appropriate agent,reagent, buffer or other material, as desired, to the second vessel,providing the vessel to a device for providing focused acoustic energyto the sample for homogenization and/or extraction, and placing thehomogenized sample in a storage location. The storage location fromwhich the sample vessel is retrieved and the storage location to whichthe homogenized sample is placed for storage may or may not be the samestorage location. Additionally, storage may or may not occur atcryogenic temperatures. Ultrasonic processes of the type employed on thesecond vessel are described in U.S. patent application Ser. No.10/777,014, the entire content of which is incorporated above byreference. According to a variation of this aspect of the invention, thesample is not transferred to a second vessel. Instead the sample vesselis used for the entire process, including the acoustic treatment of thesample, and/or other downstream processes, including centrifugation forspinning down particulates in the sample.

Additional systems, methods, devices, features and advantages of theinvention will be discussed below with respect to the illustrativeembodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be better understood by referring to the followingdescription of illustrative embodiments, taken in conjunction with theaccompanying drawings, in which like reference designations refer tolike components and depictions components are not necessarily drawn toscale.

FIG. 1 is a schematic flow diagram of a sample preparation process usinga vessel including a portion flexible enough to deform nondestructivelyin response to a mechanical impact sufficient to fragment a samplecontained within the vessel, according to an illustrative embodiment ofthe invention.

FIG. 2A is a schematic flow diagram of a sample preparation processusing a vessel of the type depicted in FIG. 1 in conjunction withanother vessel for further processing of the sample, according to oneillustrative embodiment of the invention.

FIG. 2B is a schematic flow diagram of a sample preparation processusing a vessel of the type depicted FIG. 1 in conjunction with anothervessel for further processing of the sample, according to anotherillustrative embodiment of the invention.

FIGS. 3A-3B depict two illustrative approaches for placing samples inand removing samples from vessels, according to various illustrativeembodiments of the invention.

FIG. 4 is a conceptual block diagram of an impact providing deviceaccording to an illustrative embodiment of the invention.

FIGS. 5A-5D show a schematic flow diagram depicting a sample preparationprocess using a mechanical impact providing device of the type depictedin FIG. 4, according to an illustrative embodiment of the invention.

FIGS. 6A-6E show a flow diagram depicting a sample preparation processemploying a vessel assembly according to an alternative illustrativeembodiment of the invention.

FIG. 7 depicts a sample preparation vessel according to one illustrativeembodiment of the invention.

FIG. 8 depicts a sample preparation vessel according to anotherillustrative embodiment of the invention.

FIG. 9 depicts a sample preparation vessel attached to a processingtube, according to one illustrative embodiment of the invention.

FIG. 10 depicts a sample preparation vessel formed on a substrateaccording to an illustrative embodiment of the invention.

FIG. 11 depicts a sample preparation vessel having one or more accordionlike sides according to another illustrative embodiment of theinvention.

FIG. 12 is a schematic flow diagram depicting automated and/or manualmethodologies by which a sample may be collected, stabilized, stored,fragmented, transferred, and/or processed according to variousillustrative embodiments of the invention.

FIG. 13 is a conceptual diagram depicting the invention being employedin a robot-assisted sample preparation/treatment system according to anillustrative embodiment of the invention.

FIGS. 14A-14D provides a conceptual flow diagram depicting a vessel ofthe invention employed in an experimental example using brewers yeast.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

As described above in summary, the invention provides, in variousembodiments, systems, methods and devices for collecting, stabilizing,pulverizing, fragmenting and/or analyzing biological and non-biologicalsample specimens. Before constituents of such a sample specimen can beeffectively analyzed, the sample specimen, preferably, is fragmentedinto a plurality of smaller specimens. Such smaller specimens can thenbe stored, analyzed, or further processed, as desired.

FIG. 1 is a flow diagram depicting a sample specimen fragmentationprocess 100 according to an illustrative embodiment of the invention. Asshown, in step 101, the process 100 provides a vessel 120. The vessel120 includes a bottom 123 and one or more side walls 124 and 125 forminga chamber 126. In the illustrative embodiment, the top 122 of the vessel120 is shown as open in steps 101 and 102. However, as shown in step103, in preferred embodiments, subsequent to introduction of a sample,such as the sample 126, the top 122 closed with a protective seal 130.The protective seal 130 may be, for example, a mating cover, snap cap,screw top or stopper sized and shaped to interfit with the top 122.However, in other illustrative embodiments, barrier 130 may include,without limitation, any suitable structure for sealing the top 122closed, including, without limitation, a structure for heat sealing,crimping, clipping, and/or gluing the top 122 closed. According to theillustrative embodiment, the interior surfaces of the vessel 120 iscoated with a material for facilitating heat sealing the top portion 122to a fitting (described in more detail below with respect to FIGS. 2A,2B, 8 and 9), into which a stopper may be screwed or otherwiseinterfitted. By way of example, the interior surfaces of the vessel 120may be coated with a layer of FEP (fluorinated ethylene propylene).

According to the illustrative embodiment, the vessel 120 in combinationwith the protective seal 130 provide a barrier for structurallyisolating the sample 110 from an external environment. According to oneillustrative feature, the vessel 120 maintains the sample 110 in asterile environment.

As shown at step 104, subsequent to loading the sample specimen 110 intothe chamber 126, the vessel 120 is installed into a mechanical impactproviding device. The mechanical impact providing device includes atleast one impact providing surface driven for example by a hammer,solenoid piston, pneumatically actuated device, hydraulically actuateddevice, gravity actuated device, or any other suitable mechanism (e.g.,the solenoids 140). As indicated by the arrows 128 a and 128 b, thesolenoids 140 impact at least a portion of the side walls 124 and 125 athigh velocity. The mechanical impact from the solenoids provides a forcesufficient to disrupt the macro-structure of the sample specimen 110,and fragment it into a plurality of specimens 150 between interiorsurfaces of the side walls 124 and 125. Illustratively, the impact forceis between about 10 Joules and about 25 Joules. In other illustrativeembodiments, the impact force may be greater than about 10 Joules, 12Joules, 14, Joules, 16 Joules, 18 Joules, 20 Joules, 22 Joules or 25Joules. The impact, in one example, reduces the size of the sample tofragments 150 of less than about 1 mm in the greatest dimension. In apreferred embodiment, the solenoids 140 may be caused to contact thevessel 120 more than one time to achieve the desired samplefragmentation. For example, the vessel 120 be contacted 1, 2, 3, 4, 5,or more than 5 times. When a sample is contacted multiple times, themagnitude of each impact force may be the same or may vary.

Although FIG. 1 conceptually shows two solenoids 140, in alternativeembodiments, the impact providing device may include a single movingportion having a first surface actuated to fragment the sample specimen110 against a stationary second surface.

According to the illustrative embodiment, the vessel 120 includes atleast one portion flexible enough to deform nondestructively (e.g.,without experiencing cracking, tearing, ripping or other degradation instructural integrity), or in some embodiments substantiallynondestructively, in response to the mechanical impact from the solenoid140. According to one illustrative feature, subsequent to the mechanicalimpact, the vessel 120 continues to maintain sufficient structuralintegrity to continue to isolate or substantially isolate the samplefrom the external environment, including the impact surfaces of thesolenoid 140. Preferably, the vessel 120 continues to maintain thesample 110 in sterile isolation and structurally separated from theexternal environment subsequent to the mechanical impact.

As shown in the illustrative embodiment of FIG. 1 at step 104, one orboth of the side walls 124 and 125 are deformable under the abovedescribed conditions. Also according to the illustrative embodiment ofFIG. 1, the sample is maintained at a temperature below about −20° C.,−30° C., −40° C., −50° C., −60° C., and/or −70° C. during application ofthe mechanical impact. In some implementations, the exposure to themechanical impact occurs with the sample at between about −80° C. andabout −196° C. According to the illustrative embodiment, the deformableportion of the vessel 120 remains nondestructably deformable at suchtemperatures. According to another feature, the vessel 120, with thebarrier 130 installed, may be substantially evacuated.

Preferably, at least the interior surfaces of the vessel 120 arefabricated from a material that is substantially inert, and thus doesnot react (e.g., chemically or biochemically react) with the sample 110.According to the illustrative embodiment, at least the deformableportion of the vessel 120 is fabricated from, or otherwise includes, aKapton™ (part number 150FN019 available from Dupont) or other polyimidefilm.

According to the illustrative embodiment, the interior surfaces of thevessel are coated with one or more agents useful in maintainingintegrity of the sample, such as DNase inhibitors, RNase inhibitors,protease inhibitors, anti-coagulants, anti-bacterial agents, anti-fungalagents, and/or chelating agents (e.g., EDTA, EGTA), glycerol, and/or thelike. In some illustrative embodiments, the interior of the vessel iscoated with a single agent, whereas in other illustrative embodiments,it is coated with multiple agents. These agent(s) may be used to coatthe interior of the vessel 120, regardless of the particular vesselmaterial and regardless of whether the vessel 120 includes a layer ofFEP or other heat-sealable material.

As shown at 105, the pulverized sample pieces 130 may be furtherprocessed or stored for further processing in the vessel 120, or asindicated by the arrow 132, may be transferred to another vessel forfurther processing. Further processing may include, without limitation,exposure to a focused acoustic energy source to cause, for example:sample cooling; heating; homogenizing, fluidizing; mixing; stirring;disrupting; increasing permeability of a component of; enhancing areaction of; sterilizing; and/or further sample fragmenting. Suchfurther processing is described in co-pending, co-owned U.S. patentapplication Ser. No. 10,777,014, the entire disclosure of which isincorporated by reference above. Further processing may also include anydetection, identification, measurement and/or other analysis performedon the sample.

According to another illustrative embodiment, the vessel 120 includes amechanical, optical, and/or electronic security feature, such as aninterlocking mechanical feature, radio frequency identification code, orbarcode configured for interacting with a corresponding security featureon the mechanical impact providing device (described below in detailwith regard to FIG. 4) and/or the acoustic energy providing devicedescribed in detail in patent application Ser. No. 10/777,0140, withoutwhich interaction, the mechanical impact providing device, and/or theacoustic energy providing device, respectively, will not function.

The exterior of the vessel 120 may also include markings, for example,by printing or other mechanism for various purposes such as bar-coding,user guidance and instruction, and to mark the preferred sample locationor impact target area for efficient processing. Exterior markings canoccur at any point during sample handling and processing, and may occurat multiple points during sample handling. Additionally, when samplehandling involves transfer of the sample between two or more differentvessels, such as discussed below with regard to FIG. 2A, the one or bothvessels may be marked. Marking can include barcodes (optical), RFID(electrical), punched holes (mechanical) or the like.

FIG. 2A is a schematic flow diagram of a sample preparation process 200using a vessel assembly 221, including a vessel 220 of the type depictedat 120 in FIG. 1, in conjunction with another vessel 205 for furtherprocessing the sample 110, according to a illustrative embodiment of theinvention. One particular non-limiting example of a vessel 205 is aborosilicate glass culture tube (screw-cap) with a round bottom, 16×100mm (Fisher, p/n 14-962-26F, Pittsburg, Pa.).

In a similar fashion to the vessel 120, the vessel 220 includes a bottom223 and one or more side walls 224 and 225 forming a chamber 226. In theillustrative embodiment, the vessel 220 includes a reversibly sealabletop 222. More particularly, the vessel includes a threaded fitting 227located within and affixed to the top 222. The vessel assembly 221 alsoincludes a mating cover or stopper 230 including threads 231 sized andshaped to interfit with the threaded fitting 227. With the stopper 230installed into threaded fitting 227, the vessel 220 provides, in someembodiments, a barrier for structurally isolating the sample 110 from anexternal environment, and optionally maintaining the sample 110 in asterile environment.

According to the illustrative embodiment of FIG. 2A, the threadedfitting 227 is heat bonded to the interior surfaces of the top 222 ofthe vessel 220. However, in alternative embodiments, the threadedfitting 227 may be clipped, crimped, shrink wrapped, glued or otherwiseaffixed within the top 222. According to the illustrative embodiment,the interior surfaces of the top 222 of the vessel 220 is coated with,for example, FEP (fluorinated ethylene propylene), for facilitating heatsealing the top portion 122 to the threaded fitting 227.

As shown at step 202, subsequent to loading the sample specimen 110 intothe chamber 226 and installing the stopper 230, the vessel 120 isinstalled into a mechanical impact providing device. As in the case ofthe process of FIG. 1, the mechanical impact providing device includesone or more solenoids 140. As indicated by the arrows 228 a and 228 b,the solenoids 140 contact at least a portion of the side walls 224 and225 at high velocity. The mechanical impact from the solenoids providesa force sufficient to disrupt the macro-structure of the sample specimen110, and fragment it into a plurality of specimens 150 between interiorsurfaces of the side walls 224 and 225.

As in the case of the vessel 120 of FIG. 1, the vessel 220 includes atleast one portion flexible enough to deform nondestructively (e.g.,without experiencing cracking, tearing, ripping or other degradation instructural integrity), or in some embodiments substantiallynondestructively, in response to the mechanical impact from thesolenoids 140. As also in the case of the vessel 120, subsequent to themechanical impact, the vessel 220 continues to maintain sufficientstructural integrity to continue to isolate or substantially isolate thesample 150 from the external environment, including the impact surfacesof the solenoids 140. Preferably, the vessel 220 continues to maintainthe sample 110 in structurally separated from the external environmentsubsequent to the mechanical impact.

As shown in the illustrative embodiment of FIG. 2A at step 202, one orboth of the side walls 224 and 225 are deformable under the abovedescribed conditions. As in the case of the illustrative embodiment ofFIG. 1, the exposure to the mechanical impact occurs at temperaturesbelow about −20° C., −30° C., −40° C., −50° C., −60° C., and/or −70° C.In some implementations, the exposure to the mechanical impact occurs atbetween about −80° C. and about −196° C. As in the case of theillustrative embodiment of FIG. 1, the deformable portion of the vessel220 remains nondestructably deformable at such temperatures. The vessel220, with the stopper 230 installed, may be substantially evacuated.

As in the illustrative vessel 120, at least the interior surfaces of thevessel 220 are fabricated from a material that is substantially inert,and thus does not react (e.g., chemically or biochemically react) withthe sample 110. Illustratively, at least the deformable portion of thevessel 220 is fabricated from, or otherwise includes, a Kapton™ (partumber 150FN019 available from n) or other polyimide film.

In a similar fashion to the illustrative vessel 120, the interiorsurfaces of the vessel 220 are coated with one or more agents useful inmaintaining integrity of the sample, such as DNase inhibitors, RNaseinhibitors, protease inhibitors, anti-coagulants, anti-bacterial agents,anti-fungal agents, chelating agents (e.g., EDTA, EGTA), glycerol and/orthe like. Single or multiple agent internal wall treatments may beemployed. These agent(s) may be used to treat the interior of the vessel220, regardless of the particular vessel material and regardless ofwhether the vessel 220 includes a layer of FEP or other heat-sealablematerial.

In step 203, the threaded plug 230 is removed from the top 222 of thevessel 220, and the second vessel 205, for example, a threadedborosilicate processing tube 205 having a threaded top 207 is screwedinto the threaded fitting 227. In step 204 and as indicated by the arrow209, the sample vessel 220 is then inverted to transfer the fragmentedsample specimens 150 into the processing tube 205. Transfer of thesample specimens 150 may occur by simply inverting the sample vessel 220with respect to the processing vessel. Additionally, the sample vessel220 may be flicked, tapped, or otherwise contacted with a mild force tofacilitate transfer of the sample specimens 150 into the processing tube205. In step 205, the vessel 220 is then unscrewed off the processingtube 205 and a cap 231 is screwed onto the processing tube 205 tomaintain the sample specimens 150 structurally separated from anexternal environment. As described above, with respect to FIG. 1, thesample tube 205 may be stored for later processing or be immediatelyexposed to the further processing.

As in the case of the vessel 120, the illustrative vessel 220 mayinclude a mechanical, optical, and/or electronic security feature, suchas an interlocking mechanical feature, radio frequency identificationcode, or barcode configured for interacting with a correspondingsecurity feature on the mechanical impact providing device (describedbelow in detail with regard to FIG. 4) and/or the acoustic energyproviding device described in detail in U.S. patent application Ser. No.10/777,0140, without which interaction, the mechanical impact providingdevice, and/or the acoustic energy providing device, respectively, willnot function.

According to one feature of the invention, the sample 110 is keptstructurally separated from an external environment during thefragmentation process. According to another advantage, the vessels 120and 220 may be a single use vessels that are discarded subsequent to thesample transfer step. According to another advantage, the impactsurfaces of the mechanical force providing device are not exposed to thesample 110, and thus require no cleaning before being used again. Thisenables a plurality of samples to be processed in series faster and moreefficiently. According to a further advantage, by maintaining the sample110 in sterile isolation and not requiring the fragmentation device tobe cleaned by the operator after each use, the likelihood of exposingthe operator to the sample is reduced. The likelihood of samplecontamination is also reduced.

Although in the illustrative embodiments of FIGS. 1 and 2A, the samplespecimens 150 are transferred, for example, to the tube 205 for furtherprocessing and/or storage, in alternative embodiments, the vessels 120or 220 containing the sample specimens 150 are employed for the furtherprocessing and/or storage, and no sample transfer steps 105, 203, 204and/or 205 occur. One factor in deciding whether to carry out thetransfer steps 105, 203, 204 and/or 205 may be whether the vessel120/220 or the vessel 205 is better suited for the particular furtherprocessing and/or storage.

As described above with respect to FIG. 1, one option for furtherprocessing is exposing the sample 150 to focused acoustic energy of thetype described above and in U.S. patent application Ser. No. 10/777,014.To accommodate such subsequent processing, in some illustrativeembodiments, the vessels 120 and/or 220 include at least a portion thatis substantially transparent to a focused acoustic wave of the typedescribed in U.S. patent application Ser. No. 10/777,014. In otherwords, such portion allows the described acoustic waves to pass withoutsignificant or preferably any substantial distortion. According to oneembodiment, the distortion, if any, is at least small enough to enablethe processing described in U.S. patent application Ser. No. 10/777,014to occur. According to one embodiment, the acoustically transmittingportion is located in the bottom 123/223 of the vessel. Whereas in otherembodiments, it is located in at least one of the side walls 124 and 125and/or 224 and 225.

FIG. 2B is a flow diagram depicting an alternative process 240 forfragmenting the sample 110 according to another illustrative embodimentof the invention. The process of FIG. 2B employs a vessel 220 and avessel 205 identical to and having the same advantages and properties ofthe vessels 220 and 205 of FIG. 2A. One difference between the process200 of FIG. 2A and the process 240 of FIG. 2B is that the process 240does not employ the stopper 230. Instead, after the sample 110 is loadedinto the chamber 226 in step 241, the vessel 205, in step 242, isscrewed into the fitting 227 to connect the vessels 205 and 220 to eachother. The fragmenting step 243 then proceeds in the same manner as thefragmenting step 202 of FIG. 2A. Similarly, the sample transfer step 244and the capping step 245 proceed in the same manner as the sampletransfer step 204 and the capping step 205, respectively, of FIG. 2A.

One advantage of the process 240 is that it eliminates the steps ofinserting and removing the stopper 230, thus eliminating a chance forsample contamination and operator exposure to the sample. Anotheradvantage is that the tube 205 may be used as a handle for inserting thevessel 220 into the impact providing device. It may also be used as ahandle for dipping the vessel 220 into a cryogenic fluid to maintain thesample 110 at a desired cryogenic temperature during processing. Afurther advantage of attaching the tube 205 prior to fragmentation isthat it provides additional volume, which reduces the increase inpressure due to the compression of the sample vessel 220 duringfragmentation.

As discussed above and as depicted in FIGS. 2A and 2B at 203 and 242,respectively, the sample vessel 220 may be interfitted with theprocessing vessel 205. The processing vessel 205 may have an agent,reagent, buffer or other relevant material placed in it prior tointerfitting with the vessel 220. Preferably, the processing tube 205includes a barrier for containing the material so that it does not spillinto the vessel 220 at an undesirable time. According to one feature, anoperator can cause the barrier to break to release the material into thesample vessel 220 or to allow the sample 120 to be transferred to theprocessing vessel 205. Such transfer may be caused to occur before,after or during fragmentation. Breaking of the barrier may be caused,for example, by the fragmenting process. Breaking of the barrier mayalternatively be caused by an operator rotating or otherwisemanipulating the relative positions of the sample 220 and processing 205vessels. In another illustrative embodiment, the operator squeezes thesample vessel 220 to decrease its volume sufficiently to create apressure against and break the barrier of the processing vessel 205. Anyother suitable method for breaking such a barrier may be employed.

The mechanism for placing a sample 110 in and removing it from a vessel,such as the vessels 120 and 220 described above, may employ any suitableapproach, including any conventional approach, such as those used forstoring and administering pharmaceuticals and blood products. FIGS. 3Aand 3B show two illustrative configurations for placing a sample 110 inand removing it from a vessel according to an illustrative embodiment ofthe invention.

More particularly, FIG. 3A shows the vessel 120 employing a single sealproviding element 302 for providing the conceptually indicatedenvironmental seal 130. As described with regard to FIGS. 1 and 2A, theseal providing element may include, without limitation, a mating cover,snap cap, screw top or stopper sized and shaped to interfit with the topof the vessel 120. Alternatively, the top of the vessel 120 may besealed, without limitation, by crimping, heat sealing, clipping, and/orgluing. In the configuration of FIG. 3A, the sample 120 is inserted andremoved by way of the single element 302. The element 302 may include aresealable diaphragm for injecting a fluid into the vessel 120 and/orfor removing the sample 110 from the vessel 120 with, for example, asyringe subsequent to, for example, fluidization, mixing or stirring, asdescribed in U.S. patent application Ser. No. 10/777,014.

FIG. 3B shows an alternative illustrative embodiment in which a vessel320 of the invention includes two ports, a loading port 302 of the typedescribed above with respect to FIG. 3A, and a transfer port 306. Thetransfer port 306 may be, for example, any conventional port employedfor transferring a fluid to a from a vessel. The configuration of FIG.3B enables a relatively large sample to be placed in the vessel 320 byway of the loading port 302, and removed subsequent to size reduction byway of the transfer port 306.

FIG. 4, is a conceptual block diagram of an impact providing device 400of the type which may be used with vessels, such as the vessels 120 and220, described above with respect to FIGS. 1-3B. For illustrativepurposes only, the vessel 220 of FIGS. 2A and 2B containing the sample110 is depicted as inserted into the device 400.

As shown, the device 400 includes a housing 402. The housing 402 ispreferably made of metals such as nickel, aluminum, stainless steel18-8, and/or copper, and includes a vertically disposed cavity 404having features of any suitable structure for receiving and stabilizingthe vessel 220. More particularly, cavity 404 of the impact providingdevice 400 may contain a holder, tubular barrel or other features foroptimally positioning the vessel 220 and/or the sample 110 with respectto the impact surfaces. The positioning feature may include asubstantially rigid outer wall for providing lateral support forretaining the sample vessel 220 vertically within the cavity 404. Thesubstantially rigid wall may include apertures for enabling the belowdiscussed solenoids to strike the vessel 220. The positioning featuremay be made, for example, from polypropylene.

The housing 402 also include two cavities 406 a and 406 b disposedhorizontally and extending in opposite directions from the verticallydisposed cavity 404. Opposing solenoids 408 a and 408 b are located inthe horizontally extending cavities 406 a and 406 b, respectively. Thesolenoids 408 a and 408 b include impact surfaces 409 a and 409 b,respectively. Although, the device 400 is shown with two movable impactsurfaces 409 a and 409 b, in alternative embodiments, one of the impactsurfaces may be stationary (e.g., being located on a wall as opposed toa solenoid) and the other impact surface may be movable.

The impact surfaces 409 a and 409 b may be made, for example, fromface-centered cubic (fcc) metals and their alloys, such as aluminum,stainless steel 18-8, copper, and nickel. The surfaces 409 a and 409 bmay be a hammer/anvil design and may be chilled to below about −20° C.,−30° C., −40° C., −50° C., −60° C., and/or −70° C. In someimplementations, the impact surfaces 409 a and 409 b are chilled tobetween about −80° C. and about −196° C. The impact surfaces 409 a and409 b may be substantially flat and parallel to each other.Alternatively, the surfaces 409 a and 409 b may be slightly angledrelative to each other. Slightly angled surfaces tend to concentrate thesample fragments 150 at the bottom of the vessel 220. This mayfacilitate the fragmentation of certain samples. In such embodiments,the angle of the impact surfaces 409 a and 409 b may be considered a“fixture” specifically designed to align the impact surfaces withrespect to the vessel 220 and/or the sample 110.

The housing 402 also includes a cavity 410 for receiving an ImmersionCooler/chiller (Thermo Neslab CC65, Portsmouth, N.H.) capable ofachieving at least about −35° C. In this embodiment, a chilled probe isinserted into the cavity 410 to cause the sample 110 to be maintained ata desired cryogenic temperature. The housing 402 may also include a port411 for receiving a supply of cryogenically cooled fluid, such as liquidNitrogen, to achieve the same result. The housing 402 may also include athermally insulated base 418 for reducing heat transfer between aworkbench and the device 400, thus enabling the device 400 to operatewith out the need for any additional insulation between it an a worksurface.

The device 400 also includes a processor for controlling operationalparameters of the solenoids 408 a and 408 b. Operational parametersinclude, for example, the speed, acceleration, and/or force with whichthe impact surfaces 409 a and 409 b of the solenoids 408 a and 408 b,respectively, are driven together. The operational parameters alsoinclude the number of times the solenoids 408 a and 408 b are driventogether. The illustrative impact providing device 400 also includes auser interface 414. The user interface 414 enables an operator to selectthe solenoid operational parameters, for example, according to theparticular sample, the desired degree of fragmentation (e.g., thedesired size of the fragmented sample fragments), and/or the future useof the pulverized fragments. The user interface 414 may also include anactuator for initiating the impact from the solenoids 408 a and 408 b.Alternatively, impact is automatically initiated by the processor 412 inresponse to detecting that the vessel 220 has been properly insertedinto the cavity 404. Thus, the device 400 may be operate in an automated(e.g., impact automatically initiated), semi-automated (e.g., operatorcan set operational parameters), or manual (e.g., user operates anactuator to initiate impact) mode.

The device 400 further includes sensors 416. As described above, thevessels 120 and 220 may include a mechanical, optical, and/or electronicsecurity feature, such as an interlocking mechanical feature, radiofrequency identification code, or barcode. Correspondingly, the sensors416 may includes one or more sensors sensing an appropriate securityfeature from the vessels 120 and 220. In response to the sensors 416detecting the appropriate security feature, the processor 412 enablesoperation of the solenoids 408 a and 408 b. However, according to theillustrative embodiment, in the absence of such detection, the processordoes not allow the solenoids 406 a and 406 b to function.

The sensors 416 may also include one or more temperature sensors fordetecting the temperature, for example, of the cryogenic chamber 410 orother portion of the housing 402.

Fragmentation within the device 400 may occur at any of a number oftemperatures, including across a range of cryogenic temperatures.Temperature can be monitored during pulverization via the sensors 416and adjusted, automatically by the processor 412. The temperature mayalso be adjusted via an operator selectable feature included in the userinterface 414.

When pulverization occurs at cryogenic temperatures, condensation mayform within the device 400. Such condensation can potentially interferewith electronics, such as the processor 412, that allow automation ofthe device 400, or with sensors within the device 400 that modulate, forexample, device temperature and/or impact force, and/or with sensorsthat scan detect security features on the vessel 220. Accordingly, theinvention, in some illustrative embodiments, circulates an inert,anhydrous gas (e.g., compressed nitrogen) throughout all or a portion ofthe housing 402 to reduce and/or prevent condensation in and around theelectronic elements and sensors.

Although the device 400 is described as employing the solenoids 408 aand 408 b, other impact mechanisms may be employed, such as, a vice,hammer, solenoid, pneumatically actuated device, hydraulically actuateddevice, gravity actuated device, or any other suitable mechanism.

FIGS. 5A-5C show a schematic flow diagram depicting a sample preparationprocess using a mechanical impact providing device 400 and a vessel 220of the type described above. In FIG. 5A, the device 500 is shown in arest state, with the solenoids 408 a and 408 b retracted. In FIG. 5B,the vessel 220 including a sample 110 is interfitted into an impact zonewithin the chamber 404 of the device 500. In FIG. 5C, as indicated bythe arrows 502 a and 502 b, the opposed solenoids 508 a and 508 b areactivated, causing the surfaces 409 a and 409 b to impact on the walls224 and 225, respectively, of the vessel 220. Illustratively, the vessel220 employs one or more of the above described features to enable thevessel to nondestructively deform and allow the sample 110 to befragmented between least a portion of the inner surfaces of the walls224 and 225 in response to the contact by the surfaces 409 a and 409 b.As described above, the fragmentation of FIG. 5C may occur at cryogenictemperatures. According to the illustrative embodiment, the solenoids408 a and 408 b may be fired one, two, or more times to achieve thedesired degree of fragmentation.

As shown in FIG. 5D, after the desired degree of fragmentation isachieved, the solenoids 408 a and 408 b retracted into the cavities 406a and 406 b to enable removal of the sample vessel 220. Although, thevessel 220 is shown in FIG. 5D as returning to its original shape, thisneed not be the case. However, as described above, the vessel 220,preferably, maintains its structural integrity during the fragmentationprocess.

FIGS. 6A-6E provide a flow diagram 600 illustrating use of a vesselassembly 601 with the mechanical impact providing device 400 accordingto another illustrative embodiment of the invention. Referring to FIGS.6A and 6B, the vessel assembly 601 includes a first vessel 602. Thevessel 602 includes any or all of the features relating tonondestructive deformation described above with regard to the vessels120 and 220. As in the case of the vessels 120 and 220, the vessel 602includes a top 604, a closed bottom 607 and one or more side walls 614and 615 forming a chamber 608.

The top 604 is open and includes threaded portion 616. The threadedportion 616 may be a fitting into which the end 604 interfits and isfastened, such as by heat shrinking, gluing, thermal bonding or anyother suitable mechanism. Alternatively, the threaded portion 616 may beformed into the vessel 602 itself A stopper 612 having a base 607interfits into the bottom end 606 of the vessel 602. As in the case ofthe threaded portion 616, the stopper 612 may be fastened within thebottom 606 of the vessel 602 by any suitable mechanism. A wall 610extends vertically from and circumscribes the base 607. The wall 610includes an inner threaded portion 611.

The vessel assembly 601 also includes a second vessel 620. Referringalso to FIG. 6C, the vessel 620 includes a bottom portion 622, whichincludes external 624 and internal 626 threads. The threads 624 and/or626 may be formed directly on the bottom portion 622 or may be providedby a suitable fitting. Referring to FIGS. 6A and 6B, in operation, asample 110 is introduced into the chamber 608 by way of the opening atthe end 604. The stopper 612 may be sized to position the sample 110 ata particular height in the chamber 608. The vessel 620 is then fittedconcentrically over the vessel 602 and rotated relative to the vessel620 to engage the inner threads 626 of the vessel 620 with the outerthreads 616 of the vessel 614. The vessel 620 is rotated relative to thevessel 602 until the threads 616 are passed and the vessel 620 slideover the vessel 602. As shown in FIG. 6B, the vessel 620 is then furtherrotated relative to the vessel 602 to cause the outer threads 624 on thevessel 620 to engage with the inner threads 611 on the wall 610. The twovessels may be rotated relative to each other until they are tightlyscrewed together. The assembly 601 may be stored and/or shipped in thisconfiguration until further processing is desired.

As shown in FIG. 6C, at the time of further processing, the vessel 620can be unscrewed from the wall 610, slid up the vessel 602 and rotatedto cause the inner threads 626 of the vessel 620 to engage with andscrew onto the outer threads 616 of the vessel 602. With the two vessels602 and 620 so engaged, the vessel 602 may be inserted into the device400 as discussed above to fragment the sample specimen 110 into aplurality of sample specimens 150. As shown in FIG. 6D, the assembly 601can then be inverted to transfer the fragmented specimens 150 into thevessel 620. As shown in FIG. 6E, the vessel 602 can then be screwed offof the vessel 620 and a screw cap 630 can be inserted onto the vessel620 to maintain the sample 150 in sterile isolation.

Advantage of the system of FIGS. 7A-6E over the system of FIGS. 2A and2B include: the total volume of the assembly 601 is less during storageand shipping than the volume of the vessels 220 and 225, mated as shownat 243 in FIG. 2B; a label need be applied to only one vessel; and thetransfer process is more rapid if pre-assembled as shown in FIG. 6B.

FIG. 7 is a diagram of a sample vessel 800 according to anotherillustrative embodiment of the invention. The vessel 800 includes one ormore of the deformable characteristics as described above. As describedabove, these characteristics are maintained over a wide range of impactforces, sample types, and temperature ranges, including in someembodiments, cryogenic temperature ranges. In one configuration, thesample vessel 800 is constructed from two sheets of a flexible material.Preferably, the material retains its flexibility and structuralintegrity at cryogenic temperatures. As discussed above, exemplarymaterials include, but are not limited to, polyimide films such asKapton™ film part number 150FN019 available from Dupont. The two sheetsof material can be sealed (e.g., heat-sealed, crimped, clipped, orglued) along a border 804 on three edges. Sealing the two sheets ofmaterial on three edges creates a pouch (vessel) with one open end 806.

The open end 806 of the vessel 800 can be closed with a cap 803 directlyor by way of a hub fitting 802. Both the hub fitting 802 and the cap 803may be formed, for example, from acetyl copolymer, polypropylene, or anysuitable polymer plastic. The hub fitting 802 may be slid into andsecured within the opening 806 of the vessel 800 by way of any suitablemechanism, including heat bonding, crimping, clipping, heat shrinking,and/or gluing. Illustratively, a substantially gastight seal is formedbetween the hub fitting 802 and the inner wall of the opening 806. Incertain embodiments, the inner wall near the opening 806 can besandwiched between the hub fitting 802 and an adhesive-lined heat-shrinkpolyolefin tubing (e.g., Raychem TAT 125) to help affix the flexiblevessel to the hub fitting 802 in a gastight manner. In otherembodiments, such tubing is not necessary. The inner surface of thevessel 800 near the opening 806 (e.g., an interior surface of the vesselin opposition to the hub fitting 802) can be coated or otherwisecomposed of a heat-sealable material. Exemplary heat-sealable materialsinclude, but are not limited to FEP. Such heat-sealable material canthen be used to attach the flexible vessel 800 directly to the hubfitting 802. Additionally, the opening 806 can have a funnel like shape,and be employed with our without the hub fitting 802. The cap 803 is,for example, threaded to screw reversibly into the hub interface 802. Invarious illustrative embodiments, the cap/plug 803 may be, for example,screw-fitted, snap-fitted, pressure-fitted, or magnetically attached.Optionally, the cap may be modified by punching a ⅜ inch through portinto the top, such as in the case of Fisher, p/n 14-962-26F, Pittsburg,Pa. According to other illustrative embodiments, the hub fitting 802 maybe sized and shaped for reversibly sealable mating with another vessel,so that the contents of the vessel 800 may be easily transferred to theother vessel for further processing. Such a configuration is discussedin further detail below with regard to FIG. 10.

FIG. 8 depicts a sample vessel 900 according to another illustrativeembodiment of the invention. As in the case of the sample vessel 800,the sample vessel 900 includes any or all of the above describednondestructive deformation characteristics. The sample vessel 900 isessentially the same as the vessel 800 except that it is formed from asingle sheet of material, folded to form an enclosed bottom edge 902 andbonded along the edges 906 and 908 to form a pouch 901, as opposed tothe two sheet configuration of the illustrative embodiment of FIG. 7.The vessel 900 includes the hub 802 and cap/plug 803 configuration ofthe vessel 800 of FIG. 8.

According to one illustrative embodiment, excluding the hub 802 and cap803, which are preferably formed from a substantially rigid material,the entire vessel 800 and 900 is formed from a material having the abovedescribed nondestructive deformation properties.

According to various illustrative embodiments, the vessels 800 and 900can be employed in place of the vessels 120 and 220 in any of the abovedescribed embodiments. For example, the vessels 800 and 900 can replacethe vessel 120 in any of the embodiments depicted in or discussed withregard to FIG. 1, and the vessel 220 in any of the embodiments depictedin or discussed with regard to FIGS. 2A and 2B, 3A and 3B, 4, 5A-5D, and6A-6E.

In one example of such a replacement, FIG. 9 shows the vessel 900 ofFIG. 8 interfitted with the processing vessel 205 as similarly depictedin FIGS. 2A and 2B at 203 and 243, respectively. As is the case with theconfigurations of FIGS. 2A and 2B, the processing vessel 205 may have anagent, reagent, buffer or other material placed in it prior tointerfitting with the vessels 800 or 900. Preferably, the processingtube 205 includes a barrier for containing the material so that it doesnot spill into the vessels 800 or 900 at an undesirable time. Accordingto one feature, an operator can cause the barrier to break to releasethe material into the sample vessel 900 or to allow the sample to betransferred to the processing vessel 205. Such transfer may be caused tooccur before, after or during fragmentation. Breaking of the barrier maybe caused, for example, by the fragmenting process. According to anotherillustrative embodiment, an operator rotates or otherwise manipulatesthe sample and processing vessels relative to each other to break thebarrier. In another embodiment, the operator squeezes the sample vessel900 to decrease its volume sufficiently to create a pressure against andbreak the barrier of the processing vessel 205.

Although FIGS. 7 and 8 are not shown in color, preferably, the pouches801 and 901 are preferably formed from an amber, gold or yellow coloredtranslucent material. The ornamental design of the vessels 800 and 900,generally, and the pouches 801 and 901, particularly, are considered tobe part of the herein described invention. The ornamental design of theassembly 1000 is also considered to be part of the herein describedinvention.

FIG. 10 depicts a sample vessel 1100 according to another illustrativeembodiment of the invention. The sample vessel includes a deformablewall 1101, formed as a blister pack on a substrate 1103 to form achamber 1104. One or more layers of a flexible material of the typediscussed above or other suitable material can be affixed by way of anysuitable mechanism along a periphery to the surface 1102 of thesubstrate 1103. Although not shown, the vessel 1100 may include one ormore reversibly sealable openings, for example, located on the substrate1103 or the deformable wall 1101 for inserting and removing a sampleform the chamber 1104. In operation, an impact providing device needonly provide an impact to the deformable wall 1101, with the substrate1103 braced against a stationary surface.

FIG. 11 shows a vessel 1125 according to another illustrative embodimentof the invention. As shown, the vessel 1125 includes a substantiallyrigid top wall 1122 and bottom wall 1124 and one or more creasedaccordion-like side walls 1126 and 1128. In response to an impact oneither or both of the top 1122 and bottom 1124 walls, the side walls1126 and 1128 fold and nondestructively collapse to enable a samplecontained within the vessel 1125 to be fragmented between the twosubstantially rigid walls 1122 and 1124. The vessel 1125 also includesat least one reversibly sealable port 1120 for introducing sample intoand/or removing a sample from the vessel 1125. The walls 1126 may bemade from any material suitable for performing under the above describedfragmentation and temperature conditions, including those materialsdisclosed herein. In other illustrative embodiments, the port 1120 maybe located anywhere on the device vessel.

FIG. 12 depicts a flow diagram of a process 1200 for collecting,stabilizing, and fragmenting a sample according to an illustrativeembodiment of the invention. The sample is initially collected at 1201.A biological or non-biological sample can be harvested and placed into asample processing vessel, such as the vessels 120, 220, 800, 900, 1100and 1125. The sample is inserted into the vessel via an open end orport, and the open end or port is then sealed (e.g., plugged or capped)so that the sample is contained within a closed, sample vessel. In someembodiments, the port is reversibly sealed. Given that many samples,particularly biological and non-biological samples for which an ultimateassay involves examination of a biological or chemical agent, aresensitive to contamination and/or degradation, the sample can then bestabilized at 1202. Stabilization may include, for example, thermaland/or chemical stabilization.

By way of example, stabilization 1202 may include subjecting the samplecontained within the sample vessel to cryogenic temperatures. Exemplarycryogenic temperatures include temperatures less than or equal to about−20° C. (e.g., placing the sample vessel into a freezer set to −20° C.,placing the sample vessel on dry ice). Further exemplary cryogenictemperatures include temperatures less than or equal to about −80° C.Other exemplary cryogenic temperatures include temperatures less than orequal to about −196° C. (e.g., placing the sample vessel into liquidnitrogen). In a preferred embodiment, the sample vessel is constructed,at least in part, from materials that nondestructively deform in amanner, such as that discussed supra, across the temperature range atwhich the sample is manipulated. Preferred materials include materialswith rapid heat transfer characteristics (e.g., materials that rapidlytransfer temperature from the external environment to the interior ofthe vessel such that a sample within the vessel rapidly reaches adesired temperature).

The stabilization step 1202 may also include chemical stabilization. Forexample, agents that inhibit degradation of sample constituents can beintroduced into the vessel, or alternatively, the vessel may be treatedwith such constituents. These agents can be added prior to,concomitantly with, or following collection of the sample into thesample vessel. Exemplary agents include, but are not limited to, DNaseinhibitors, RNase inhibitors, protease inhibitors, anti-coagulants,anti-bacterial agents, anti-fungal agents, chelating agents (e.g., EDTA,EGTA), glycerol, and/or the like.

At step 1203, following stabilization, the sample may be stored for someperiod of time prior to pulverization. Prior to storage, the samplevessel containing the sample may be marked with a label or bar code tofacilitate later identification. The sample can be shipped and stored ata separate location or can be stored at the site of sample collection.Depending on the particular sample, storage illustratively occurs atcryogenic temperatures ranging from about −20° C. to about −196° C.However, the invention contemplates that for certain samples and certainapplications, storage can occur at temperatures greater than −20° C.,for example, storage can occur at room temperature.

Following stabilization at 1202, rather than be stored at 1203, thesample may be pulverized/fragmented at 1204. As described above, thefragmenting step 1204 may include contacting a sample vessel containinga sample with a mechanical force, sufficient to achieve the desiredfragmentation. According to the various illustrative embodiments, thesample vessel includes at least a portion that is nondestructivelydeformable to enable the mechanical force to be transferred to thesample through the vessel while maintaining the sample in isolation fromthe external environment, generally, and the impact surface or surfaces,particularly. The invention thus provides non-contact, mechanicalfragmentation of the sample contained in the sample vessel.

As described supra, the mechanical impact force to the sample may beprovided by suitable mechanism, including for example, a hammer.However, in a preferred embodiment, a mechanical impact providingdevice, such as that described with regard to FIGS. 4 and 5.

As depicted in FIG. 12, the fragmentation step 1204 (as described in theprevious two paragraphs) may occur following some period of storage.Note that this period of storage can vary based on the particularsample, its intended use, etc. Exemplary storage periods include shortterm storage for minutes (e.g., less than or equal to 30 minutes) orhours (e.g., less than or equal to 1, 2, 5, 10, or 12 hours). Furtherexemplary storage periods include overnight storage or storage for about1-3 days, 3-5 days, 1 week, 2 weeks, 4 weeks, or greater than 4 weeks.Still further exemplary storage periods include long range storage for3-6 months, 6-12 months, 1-2 years, greater than 2 years, 2-10 years, orlonger.

Following fragmentation in step 1204, the sample vessel may be furthermarked, for example, to provide information relevant fragmentation, suchas the degree to which the sample has been fragmented. Next, the samplemay be stored in step 1206 in a similar fashion to that described abovewith respect to step 1206, transferred in step 1205 for furtherprocessing in step 1207, or further processed in step 1207 withouttransferring the sample out of the original sample vessel.

Transferring to a processing vessel at 1205 can occur by directlyattaching the processing vessel to the sample vessel, inverting thesample vessel, and optionally flicking or tapping the sample vessel sothat substantially all of the sample is transferred from the samplevessel into the processing vessel. Alternatively, sample transfer 1205can occur without directly attaching the processing vessel to the samplevessel. Transfer 1205 may occur at cryogenic temperatures in the rangesdiscussed above, for example, by pre-chilling the processing vesselprior to sample transfer.

In certain embodiments, the sample transfer 1205 is facilitated byaddition of a liquid to either the sample vessel or the processingvessel. The liquid may include a buffer or solvent and may optionallyinclude agents that prevent degradation of sample, such as DNaseinhibitors, RNase inhibitors, protease inhibitors, anti-coagulants,anti-bacterial agents, anti-fungal agents, chelating agents (e.g., EDTA,EGTA), glycerol and/or the like. Such agents may be particularly usefulif the sample thaws during sample transfer or if further processing ofthe sample is not to occur at cryogenic temperatures. When a liquid isused to facilitate sample transfer 1205, the liquid can be added to thesample vessel prior to transfer of any material to the processingvessel. Alternatively, the liquid can be added to the sample vesselafter the sample is transferred to the processing vessel to ensure thatsubstantially all of the sample is transferred to the processing vessel.As indicated in FIG. 12, the sample transfer step 1205 may also occurafter a period of storage at step 1206

Further processing at 1207 may include, without limitation, cooling;heating; fluidizing, mixing, stirring, disrupting, increasingpermeability of a component of, enhancing a reaction of, sterilizing,and/or further fragmenting the sample. Such further processing isdescribed in co-pending, co-owned U.S. patent application Ser. No.10/777,014, entitled Apparatus and Methods for Controlling SonicTreatment,” the entire disclosure of which is incorporated above byreference. Further processing may also include, any detection,measurement, identification or other analysis performed on the sample.

According to an additional illustrative embodiment, the invention isdirected to systems and methods for processing a sample employing anautomated vessel handling robot. Such a robot may perform any or all ofthe vessel handling steps of FIG. 12. FIG. 13 shows another diagram 1300illustrative of an approach using an vessel handling robot 1302. Asshown in FIG. 13, the vessel handling robot 1302 may be employed forretrieving a sample vessel of the type described herein from a storagelocation 1304, providing it to an impact providing device 1306 forfragmenting the sample, inverting the sample vessel to transfer thefragmented sample into a second vessel at 1308, adding an appropriateagent, reagent, buffer or other material, as desired, to the secondvessel at 1310, providing the vessel to a device for treating the samplewith focused acoustic energy, for example, for homogenization and/orextraction at step 1312, and placing the homogenized sample in a storagelocation at 1314. The storage location from which the sample vessel isretrieved at 1304 and the storage location to which the homogenizedsample is placed at 1314 may or may not be the same storage location.Additionally, storage may or may occur at any suitable temperatureincluding cryogenic and non-cryogenic temperatures. Ultrasonic processesof the type employed on the second vessel are described in U.S. patentapplication Ser. No. 10/777,014, the entire content of which isincorporated above by reference. According to a variation of this aspectof the invention, the sample is not transferred to a second vessel at1302. Instead, the sample vessel is used for the entire process, andtransferred directly from 1306 to 1310. Additionally, in someillustrative embodiments, the acoustic treatment of 1312 may be skipped.

According to a further feature, the throughput of the above describedautomated process may be increased by having multiple samples processedin a batch mode. For example, 96 samples may be fragmented eitherserially or in parallel. The samples may be transferred from process toprocess and even from sample vessels to further processing vessels inbatch mode. Additionally, the sample vessels may include dimples orindents on an interior surface to localize the sample. Samples may varyin size, and in one example are about 5 mg. As discussed above, thesamples may be maintained at or above cryogenic temperatures, dependingon the sample.

The methods and systems of the invention are useful in any of a numberof applications in which a sample is to be fragmented to smaller piecesof the specimen prior to further analysis of the sample in whole or inpart. The methods, systems and devices of the invention allow non-directcontact fragmentation of a sample and furthermore allow fragmentationunder conditions that prevent degradation of constituents of the sample.The invention can be used to collect and process a range of biologicaland non-biological samples. Accordingly, the invention has a wide arrayof applications, for example, in the fields of medical research, medicaldiagnostics, agricultural research, food safety, bio- and chemicalhazard management and safety, and the like.

By way of example, many assays, including diagnostic assays, are basedon the detection of nucleic acids or proteins contained within a sample.Such assays include, but are not limited to, PCR, RT-PCR, hybridizationto nucleic acid or protein-based micro-arrays, and immunohistochemistry.Reproducible analysis using these assays requires “clean” startingsamples (e.g., samples that have not been contaminated or exposed todegradation agents) that have been processed to allow access to theirconstituent elements (e.g., samples that have been sufficientlyfragmented and homogenized to allow access to nucleic acids, proteins,and small molecule constituents contained within the sample).

By way of a more particular example of the medical application of theinvention, it may be used to process pathological and/ornon-pathological tissue samples harvested from a patient. Such samplesinclude, but are not limited to, putative tumor samples taking during abiopsy. Prior to analysis of the nucleic acid, protein, or smallmolecule constituents of the tissue sample, the sample can be collected,stabilized, and pulverized using the above described methods, systemsand devices of the invention.

By way of a further non-medical example, the invention can be used inthe food industry. One issue encountered in the food industry is that ofpossible bacterial, viral, fungal, prion and/or chemical contamination.Presence of such possible contaminants within a sample of a food productand/or ingredient may be assayed using available biochemical and/ormolecular biological tests. Prior to biochemical and/or molecularanalysis, a given sample of food may be collected, stabilized to preventdegradation of the bacterial and/or chemical agents that one wishes todetect, and pulverized to yield smaller specimens amenable to furtherprocessing and analysis, according to the above described illustrativeembodiments of the invention.

By way of another example, the methods, systems and devices of theinvention may be used in industries such as the petro-chemical industry.Analysis of the mineral composition of particular solid samples at themolecular, chemical, and/or atomic level are useful in this industry foridentifying, for example, commercially advantageous lodes, outcroppings,and/or drilling sites. Solid samples such as rocks and mineral depositsare often too large to be directly analyzed using molecular and/orchemical tools. Accordingly, such samples can be collected, stabilized,and fragment to yield smaller specimens amenable to further processingand analysis, according to the above described illustrative embodimentof the invention.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the invention, and are not intended to limit the scope of invention.

Example A

Referring to FIGS. 14A-14D, brewers yeast was aliquoted into a vesselformed from a 2 mil Kapton™ film part number 150FN019, available fromDupont, similar to those depicted in FIGS. 7, 8, and 9, however, withouta rigid hub and/or cap. As shown in FIG. 14A, dry yeast spores wereinserted into the vessel, the mass of the spores was 35 mg. As shown inFIG. 14B, the open top of the vessel was folded over and held in placewith a large paper clip. The air space in the pouch was minimized. Thespores were located at the bottom of the vessel. As shown in FIG. 14C,depending on size, the spores may be located differently in the vessel.The vessel was then inserted into a manual, vertical hammer/anvildevice. The hammer was raised released three times. As shown in FIG.14D, the resultant yeast spores were visibly cracked and opened. As alsoshown in FIG. 14D, the vessel was opened and the fragmented spores weretransferred to a test tube. A PBS salt solution was then added. Theresultant yeast particles were and PBS salt solution was then subjectedto a focused acoustic extraction, such as that disclosed in U.S. patentapplication Ser. No. 10/777,014, the entire content of which isincorporated above by reference. The supernatant was visibly cloudyafter the ultrasonic extraction, indicating lysis and extraction ofbiomolecules. In contrast, exposing a control to the same acousticextraction process, without first fragmenting/pre-cracking the spores,resulted in an essentially clear solution, indicating no lysis orbiomolecule extraction. One advantage of the fragmentation process ofthe invention is that it avoids the using any enzymes or detergents. Theprocess of the invention is also rapid and may be run at cryogenictemperatures.

Example B

Referring generally to the device shown FIGS. 1 and 8, a sample vesselwas made by heat-sealing a 2 mil Kapton™ film part number 150FN019available from Dupont on three sides. The dimension of the two sealedsides was about 2.5 inches and the width was about 1.5 inch. A tissuesample of 1 gram of fresh beefsteak was placed into the vessel. The openend of the vessel was clamped and the vessel with the tissue was placedinto a −80° C. freezer for 30 minutes. The vessel with the sample nowfrozen was removed and placed on a previously chilled metal plate.Another metal plate was placed over the tissue area of the vessel. Aurethane 5-pound hammer was struck once to pulverize the chilled tissuesample into smaller pieces. The vessel retained flexibility and did nottear. The pulverized pieces were transferred to a borosilicate 16 mm×100mm culture tube for further processing and 2 ml of distilled water wasadded. The culture tube with the pulverized tissue and distilled waterwas homogenized using Covaris E200 system (Woburn, Mass.) for 30seconds. The Covaris E200 is described in relevant part in U.S. patentapplication Ser. No. 10/777,014 incorporated above by reference.

Example C

Referring generally to FIGS. 2 and 9, a sample vessel was constructedfrom a modified polypropylene screw cap that fits a 16 mm×100 mm culturetube. A ⅜^(th) inch opening was punched through the top. A pouch formedfrom Kapton™ (part number 150FN019 available from) film was affixed tothe polypropylene screw cap with 1 inch of heat-shrink tubing. A sampleof previously-frozen liver was inserted through the opening in the screwcap into the pouch and the cap was sealed. The loaded vessel and samplewere placed on dry ice. The vessel was laid on a metal plate previouslychilled to −80° C. and a block of Dehin (1 inch×1 inch×3 inch) wasplaced over the tissue area of the vessel. A metal hammer was used toimpact the block thereby transferring the impact through the flexibleKapton™ film and to the brittle liver sample. The vessel containing thefragmented sample was screw attached to a previously chilledborosilicate tube in an end-to-end fashion. The sample vessel was theninverted to transfer the contents to the borosilicate tube. The samplevessel was removed and 2 ml of GITC-based RNA stabilization buffer wasadded to the borosilicate tube. The sample contained within theborosilicate tube was loaded into Covaris E200 system and thoroughlyhomogenized within 15 seconds.

Those skilled in the art will know or be able to ascertain using no morethan routine experimentation, many equivalents to the embodiments andpractices described herein. Accordingly, it will be understood that theinvention is not to be limited to the illustrative embodiments disclosedherein, but is to be understood from the following claims. It should benoted that any operable combinations between any of the systems,methods, and devices described herein are considered to be patentablesubject matter, including any such operable combinations involving thedisclosure of U.S. patent application Ser. No. 10/777,014, thedisclosure of which is above incorporated by reference.

1-5. (canceled)
 6. A vessel for containing a sample, the vesselcomprising, a reversibly sealable chamber for containing the sample, aflexible portion of the reversibly sealable chamber, the flexibleportion of the chamber being flexible enough to allow the vessel todeform nondestructively in response to a mechanical impact sufficient tofragment the sample into a plurality of sample fragments, and having animpact energy transfer of greater than or equal to about 10 Joules.
 7. Avessel for containing a sample, the vessel comprising, a reversiblysealable chamber for containing the sample, a flexible portion of thereversibly sealable chamber, the flexible portion of the chamber beingflexible enough to allow the vessel to deform nondestructively inresponse to a mechanical impact sufficient to fragment the sample into aplurality of sample fragments, and a port for reversibly engaging with asecond vessel to enable transfer of material between the sample vesseland the second vessel while maintaining a barrier between the sample andan external environment.
 8. The vessel of claim 6, wherein the flexibleportion is formed from a material that remains nondestructivelydeformable at temperatures less than about −40° C.
 9. (canceled)
 10. Thevessel of claim 6, wherein the flexible portion is formed from amaterial that remains nondestructively deformable at temperaturesbetween about −40° C. and about −196° C.
 11. (canceled)
 12. (canceled)13. The vessel of claim 6, wherein the flexible portion is formed from amaterial that does not crack, tear, or rip when exposed to themechanical impact.
 14. The vessel of claim 6, wherein the flexibleportion is formed from a material that deforms nondestructively inresponse to the mechanical impact providing an impact energy transfer ofat least about 14 Joules. 15-17. (canceled)
 18. The vessel of claim 6,wherein at least a portion of the vessel that is contacted by themechanical impact has a thickness of between about 0.5 and about 5 mil.19-26. (canceled)
 27. The vessel of claim 6 including a reversiblysealable port, which, in combination with the reversibly sealablechamber, forms a barrier between the sample and an external environment.28. The vessel of claim 6, wherein at least the flexible portion isformed at least in part from one of: polyimide, polysulfone, fluorinatedpolymer, or a liquid crystal polymer. 29-32. (canceled)
 33. The vesselof claim 6, wherein the chamber is defined by two pieces of a filmmaterial bonded together along their peripheries and reversibly sealed.34. The vessel of claim 6, wherein the chamber is defined by a singlepiece of a film material folded and bonded together along a portion ofits periphery and reversibly sealed in a substantially air tightfashion.
 35. The vessel of claim 27, wherein the reversibly sealableport is sized and shaped for sealably mating with a second vessel. 36.The vessel of claim 6, further including a security feature forinteracting with a corresponding security feature on a device forproviding the mechanical impact.
 37. The vessel of claim 6, wherein aninternal surface of the chamber is treated with at least one of DNaseinhibitors, RNase inhibitors, protease inhibitors, anti-coagulants,anti-bacterial agents, anti-fungal agents, chelating agents, andglycerol. 38-55. (canceled)
 56. The vessel of claim 7, wherein theflexible portion is formed from a material that remains nondestructivelydeformable at temperatures less than about −40° C.
 57. The vessel ofclaim 7, wherein the flexible portion is formed from a material thatremains nondestructively deformable at temperatures between about −40°C. and about −196° C.
 58. The vessel of claim 7, wherein the flexibleportion is formed from a material that does not crack, tear, or rip whenexposed to the mechanical impact.
 59. The vessel of claim 7, wherein theflexible portion is formed from a material that deforms nondestructivelyin response to an impact energy transfer of at least about 14 Joules.60. The vessel of claim 7, wherein at least a portion of the vessel thatis contacted by the mechanical impact has a thickness of between about0.5 and about 5 mil.
 61. The vessel of claim 7, wherein at least theflexible portion is formed at least in part from one of: polyimide,polysulfone, fluorinated polymer, or a liquid crystal polymer.
 62. Thevessel of claim 7, including a security feature for interacting with acorresponding security feature on a device for providing the mechanicalimpact.
 63. The vessel of claim 7, wherein an internal surface of thestructural barrier is treated with at least one of DNase inhibitors,RNase inhibitors, protease inhibitors, anti-coagulants, anti-bacterialagents, anti-fungal agents, chelating agents, and glycerol.